Phosphatidylinositol-4,5-bisphosphate (PIP2) modulates the actin cytoskeleton, cell migration, invasion and signaling by binding effectors that regulate cytoskeletal dynamics and receptor trafficking. Yet, the mechanism of how PIP2 is generated to regulate these processes is poorly understood. Phosphatidylinositol phosphate kinases (PIPKs) that synthesize PIP2 specifically associate with PIP2 effectors. PIP2 then modulates the proteins and enzymes associated with the PIPK. PIPKI? regulates growth factor stimulated directional cell migration, vesicular trafficking of adhesion receptors and invasion. Here, we show that PIPKI? and PIP2 are required for establishment of cell polarity required for directional cell migration and invasion. In addition, we have discovered a novel signaling nexus that may explain how PIP2 mechanistically regulates many key effectors in adhesion, migration and invasion. Hypothesis: PIPKI? controls key aspects of tumor cell migration and invasion by controlling the assembly of cytoskeletal components and the trafficking of adhesion receptors. Interactions between PIPKI?, the exocyst and IQGAP1 function to integrate and specify the PIP2 generation and signaling required for growth factor stimulated directional cell migration, adhesion and invasion. In vivo the PIPKIg signaling nexus plays a key role in tumor cell intravasation and extravasation, processes required for tumor cell metastasis. Aim 1. The role of PIPKI?i2, the exocyst, and ?1-integrin trafficking in directional cell migration will be investigated. Regulation of integrin trafficking by PIPKI? and the exocyst will be defined with an emphasis on regulation by PIP2, the protein-protein interactions and cell polarity pathways, roles of small G-proteins, and specificity toward integrins. We will determine if a PIPKI?i2 interaction with talin acts as a tether for targeting the PIPKI?i2/?1-integrin/exocyst complex during directional cell migration. Aim 2. Define the mechanism for PIPKI? and IQGAP1 regulation of directional migration. We will study the IQGAP1/PIPKI? interaction and define how signals modulate this nexus. We will reveal the mechanism for PIPKI? control of IQGAP1 association with membrane. We will determine if and how PIPKI? modulates the association or activity of known IQGAP1 interactors, such as small G-proteins and the exocyst. Aim 3. Define the role of the PIPKI?, IQGAP1 and exocyst in cell invasion. The role of PIPKI?, the exocyst and IQGAP1 in formation of invadopodia will be revealed. An emphasis will be on targeting of key proteins required for invasion, such as IQGAP1, the exocyst, ?1-integrin and MMPs to the invadopodia in a 3D matrix environment. Aim 4. Determine if PIPKI?, the exocyst and IQGAP1 are required for extravasation and metastasis. Highly metastatic human breast cell lines will be engineered to over or under express PIPKI?, IQGAP1 or Exo70. We will use a well-established mouse xenograft model to transplant engineered cells into the vasculature. The development and number of metastases will be used to quantify extravasation. PUBLIC HEALTH RELEVANCE: Cancer deaths occur because tumor cells migrate from the initial tumor site to other organs ultimately resulting in organ failure. This process predominatel occurs via the vasculature. In order for tumor cells to leave the primary tumor and enter the vasculature they must first alter their cellular phenotype. The goal of the proposed research is to identify the relevant enzymes and proteins that contribute to the phenotypic changes resulting in enhanced migration and invasion, so that clinical therapies can be developed to target these critical molecules.